Proteolytic enzymes have been implicated in all stages of cancer progression including growth, angiogenesis, and metastasis. It is the ability of malignant cells to invade and metastasize that greatly complicates the treatment of cancer in humans. Therefore, inhibition and modulation of cancer associated proteases is an attractive treatment avenue. Protease inhibitors are potentially much less toxic with fewer side effects than most of the chemotherapeutic agents now in use, and may have equal or superior efficacy for disease control. This Project is aimed at evaluating existing protease inhibitors and developing new ones as supplements to existing treatments for cancer growth, angiogenesis, and metastasis. Two parallel approaches will be used. First, small molecules obtained from industrial and university collaborators will be screened for activity against proteases identified by the Core B section of this collaborative agreement. In addition, structure-based drug design techniques, including directed combinatorial chemistry, will be employed to identify potential new lead compounds using structural analysis of selected proteases known to be involved in cancer. Secondly, macromolecular protease inhibitors will be screened for activity against identified proteases. Protein engineering of these macromolecules, both directed and random, will be employed to modulate binding efficiencies and increase specificity. These modified macromolecular inhibitors will then be assayed in cell culture and animal model systems in Projects 2 and 3. This work can form the basis for entirely novel treatment modalities, using new drugs or drugs developed for other purposes. By interacting closely with Core D compounds with useful pharmacological properties can be studied early in the drug development process. We can be very optimistic that many of the aims of Project 1 can be met within the time frame of this program project. The protease discovery and production project is an area in which we have had extensive experience and prior success. It is very likely that new leads and existing compounds will be effective and potent inhibitors for several key proteases. The macromolecular inhibitors that are developed will provide powerful new reagents for aiding in dissecting the role of proteases in the various stages of cancer. Analysis of target proteases using structure-based drug design techniques and protein engineering can allow a new generation of anticancer drugs to be generated, thus augmenting existing treatment regimens.